Smokeless tobacco products, such as moist snuff repetitively placed in the mouth, either as a pinch of tobacco or in a tea bag-like sachet, are becoming increasingly popular in the United States. These products are accepted causes of oral cavity cancer. Tobacco-specific nitrosamines such as N2-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are recognized as likely causative agents for oral cancer in smokeless tobacco users, but previous animal testing data in support of this concept have been somewhat equivocal. We have recently completed a carcinogenicity study in rats of (S)-NNN, the major form of NNN present in smokeless tobacco products. The results of this study demonstrated for the first time that (S)- NNN is a remarkably strong oral cavity carcinogen in the rat, inducing a 100% incidence of oral cavity tumors, and a total of 91 oral cavity tumors in the 20 rats which came to necropsy. Thus, (S)-NNN is the only strong oral cavity carcinogen identified in smokeless tobacco products. A high incidence of esophageal tumors was also observed. These results provide mechanistic support for the epidemiologic observations that smokeless tobacco products cause oral cancer, and create a new direction for assessing the potential carcinogenicity of smokeless tobacco products, highly relevant to regulation by the Food and Drug Administration under the Family Smoking Prevention and Tobacco Control Act. Therefore, the Specific Aims of this Competitive Revision Application are: 1. Quantify levels of (S)-NNN in traditional and emerging smokeless tobacco products. 2. Quantify levels of (S)-NNN and (S)-NNN-N-glucuronide in the urine of users of smokeless tobacco products. 3. Develop a new urinary biomarker for assessing NNN uptake in humans. The results of this study will be crucial in defining the risks associated with smokeless tobacco use and will inform FDA regulation of NNN in smokeless tobacco products. This application is a Competitive Revision to R37 CA-81301-14, entitled Metabolism of Carcinogenic Tobacco-Specific Nitrosamines, which does not include the aims listed above. PUBLIC HEALTH RELEVANCE: This project is highly relevant to public health because the use of smokeless tobacco products is increasing in the United States, and these products are accepted causes of oral cavity cancer. The results will provide critical information on levels of the oral cavity carcinogen (S)-NNN in these products, and on its uptake by smokeless tobacco users. These new data are critical to FDA's mission in tobacco product regulation.